More than 50% of organic carbon on earth is found in the cell walls of plants. Plant cell walls consist mainly of three compounds: cellulose, hemicellulose, and lignin. Collectively these compounds are called “lignocellulose,” and they represent a potential source of sugars and other organic molecules for fermentation to ethanol or to other high-value products.
The conversion of lignocellulosic biomass to ethanol has become a key feature of emerging energy policies due to the environmentally favorable and sustainable nature of cellulosic ethanol. There are several technologies being developed for cellulose conversion. Of interest here is a method by which lignocellulosic biomass is subjected to a pretreatment that increases its susceptibility to hydrolytic enzymes, followed by enzymatic hydrolysis to sugars and the fermentation of those sugars to ethanol or other high-value organic molecules (e.g. butanol). Common pretreatment methods include dilute acid steam explosion (U.S. Pat. No. 4,461,648), ammonia freeze explosion (AFEX; Holtzapple et al., 1991), and organosolv extraction (U.S. Pat. No. 4,409,032). Hydrolysis and fermentation systems may be either separate (sequential hydrolysis and fermentation; SHF) or coincident (simultaneous saccharification and fermentation; SSF). In all instances, the hemicellulose and cellulose are broken down to sugars that may be fermented, while the lignin becomes separated and may be used either as a solid fuel or as a source for other organic molecules.
The choice of enzymes for conversion of pretreated lignocellulosic biomass to sugars is highly dependent upon the pretreatment method. Dilute acid steam explosion results in significant chemical hydrolysis of the hemicellulose, making enzymes for the conversion of hemicellulose to sugars less relevant to the process. In contrast, AFEX and organosolv extraction both leave hemicellulose and cellulose largely intact. Organosolv extraction, unlike dilute acid steam explosion or AFEX removes a significant portion of the lignin from substrate. In all instances, the primary target for enzymatic hydrolysis is the cellulose, which is converted to sugars using a combination of cellulase enzymes.
There are two principle types of cellulase enzymes: endoglucanases, which cleave glycosidic bonds in the middle of cellulose chains, and in doing so create new chain ends, and cellobiohydrolases, which cleave short oligosaccharides from the ends of cellulose chains. Glucosidases digest short oligosaccharides into monosaccharides. These three enzyme components thus act synergistically to create an efficient cellulolytic enzyme system. Most cellulases have a similar modular structure, which consists of a catalytic domain, linker peptide and a carbohydrate-binding module (CBM).
Fungal CBMs (Family 1) consist of a small wedge-shaped fold. Three solvent exposed hydrophobic (aromatic) residues lie on one surface of this fold and constitute the cellulose binding surface (Kraulis et al., 1989; Mattinen et al., 1997). These aromatic residues form van der Waals interactions and aromatic ring polarization interactions with glucose rings in the cellulose polymer (Mattinen et al., 1997; Reinikainen et al., 1992, Tormo et al., 1996). CBMs are reportedly involved in lignin binding. For example, removal of the CBM from Trichoderma Cel7A essentially eliminates binding to alkali extracted lignin and to residual lignin prepared by enzyme hydrolysis (Palonen et al., 2004).
Catalytic domains are also reportedly involved in binding lignin. Cel7B from Humicola sp., which does not possess a CBM, is bound extensively by lignin (Berlin et al., 2005b). Similarly Trichoderma Cel5A core, devoid of a CBM, binds alkali extracted lignin but to a lesser extent than does the full-length protein (Palonen et al., 2004).
Naturally-occurring linker peptides in cellulase and hemicellulase enzymes, whether from bacterial or fungal sources, vary from 6-59 amino acids in length. These peptides are similar in their chemical properties and amino acid composition, if not their specific sequences, with the amino acids serine, threonine, and proline accounting for more than 50% of the amino acids in the linker peptide (reviewed in Gilkes et al. (1991). Serine and threonine residues may be modified with O-linked glycans, which, in fungi, are predominantly mannose (Fägerstam et al., 1984). Linkers also contain several charged residues of a common type, either all negative (such as Glu or Asp) or all positive (such as Lys, Arg or His).
Linker peptides maintain the spatial orientation of the catalytic domain relative to the CBM. Shen et al. (1991) demonstrated that deleting the linker peptide altered the relative orientation of the catalytic domain and CBM of Cellulomonas fimi CenA without altering the tertiary structure of either domain. Effects of the linker peptide on the global conformation of Cel45A from Humicola insolens have been studied by small angle x-ray scattering (SAXS) (Receveur et al., 2002) and dynamic light scattering (Boisset et al., 1995). These investigators concluded that the linker peptide is an extended yet flexible structure and that glycosylation of the linker peptide favours more extended conformations, altering the relative positioning of the catalytic domain and CBM. Similarly, analysis of Cex from Cellulomonas fimi by NMR indicated that glycosylation of the linker peptide sterically constrains its flexibility, resulting in a more extended conformation and increasing the mean separation of the catalytic domain and CBM (Shen et al., 1991). A Humicola Cel6A-Cel6B chimeric double cellulase analyzed by SAXS showed that the linker peptide was flexible, adopting a compact rather than an extended conformation (von Ossowski et al., 2005). The authors suggested that the compact structure may be related to low levels of O-linked glycosylation in the Cel6A and Cel6B linker peptides.
Linker peptides modulate the binding of glycosyl hydrolases to cellulose and their enzymatic activity. Removing linker peptide from the Cellulomonas fimi CenA cellulase not only altered its structure, but reduced its catalytic efficiency. Although adsorption to cellulose was not affected, removal of the linker peptide impaired desorption of the bound enzyme from the crystalline cellulose substrate Avicel. Partial deletion of the linker peptide from Trichoderma Cel7A reportedly reduces its binding capacity on crystalline cellulose, while effects on catalytic activity were negligible (Srisodsuk et al., 1993). Complete removal of the Cel7A linker peptide reduced cellulolytic activity by 50%. These studies utilized Avicel and bacterial cellulose, essentially pure cellulosic substrates. However, these substrates do not fully represent the heterogeneity of lignocelluloses generated by commercial pretreatment processes, in particular because they do not contain lignin.
Variants of Trichoderma reesei Cel7A and Cel6A to improve thermostability have been reported (U.S. Pat. No. 7,375,197; WO 2005/028636; U. S. Publication No. 2007/0173431; Publication No. 2008/167214; WO 2006/074005; Publication No. 2006/0205042; U.S. Pat. No. 7,348,168; WO 2008/025164). In particular, substitution of the serine at position 413 in T. reesei Cel6A with a proline, or substitution of the amino acid at the equivalent to position 413 with a proline in other Family 6 cellulases confers increased thermostability (WO 2008/025164). Mutations at the equivalent of positions 103, 134, 136, 186, 365 and 410 within the catalytic domain of T. reesei Cel6A and other Family 6 cellulases have been shown to lead to reduce inhibition by glucose (U. S. Publication No. 2009-0186381). Variants with resistance to proteases and to surfactants for detergent formulations have been created for textile applications (WO 99/01544; WO 94/07998; and U.S. Pat. No. 6,114,296).
In most instances, mutations are specifically directed to the catalytic domain of the enzyme. In some instances the carbohydrate binding module has been targeted. Only in a few instances has the linker peptide been identified as playing a critical role or as a target for modification. The linker peptide of the Humicola family 45 endoglucanase was modified to reduce proteolysis (WO 94/07998; U.S. Pat. No. 6,114,296) and the linker peptide of the Trichoderma Cel7A was modified to promote thermostability (U.S. Pat. No. 7,375,197). Otherwise, the linker peptide region is typically ignored as a specific target for enzyme improvement.
The negative effects of lignin on cellulase enzyme systems are well documented. Removal of lignin from hardwood (aspen) was shown to increase sugar yield by enzymatic hydrolysis (Kong et al., 1992). Similarly, removal of lignin from softwood (Douglas fir) was shown to improve enzymatic hydrolysis of the cellulose, an effect attributed to improved accessibility of the enzymes to the cellulose (Mooney et al., 1998). Other groups have demonstrated that cellulases purified from Trichoderma reesei bind to isolated lignin (Chernoglazov et al., 1988) and have speculated on the role of the different binding domains in the enzyme-lignin interaction (Palonen et al., 2004). Binding to lignin and inactivation of Trichoderma reesei cellulases has been observed when lignin is added back to a pure cellulose system (Escoffier et al., 1991). Only in one instance was lignin reported to not have any significant effect on cellulases (Meunier-Goddik and Penner, 1999). Other reports suggest that some hemicellulases may be resistant to, and even activated by, lignin and lignin breakdown products (Kaya et al., 2000). Thus, it is generally recognized that lignin is a serious limitation to enzymatic hydrolysis of cellulose.
The development of lignin resistant cellulases represents a large hurdle in the commercialization of cellulose conversion to soluble sugars including glucose for the production of ethanol and other products. However, the lignin resistant enzymes must preserve their cellulose binding affinity and native cellulolytic activity. A variety of methods have been suggested to reduce the negative impact of lignin on the cellulase system. Non-specific binding proteins (e.g. bovine serum albumin; BSA) have been shown to block interactions between cellulases and lignin surfaces (Yang and Wyman, 2006; U.S. Publication No. 2004/0185542A1; U.S. Publication No. 2006/088922A1; WO2005/024037A2). Other chemical blocking agents and surfactants have been shown to have a similar effect (Tu et al., 2007; U.S. Pat. No. 7,354,743).
Recently, modified cellulases exhibiting reduced interactions with, or inactivation by, lignin have been reported. For example, WO2010/012102 reports that mutations at the equivalent of positions 129, 322, 363, 365 and 410 within the catalytic domain of T. reesei Cel6A (TrCel6A) and other Family 6 cellulases results in increased hydrolytic activity in the presence of lignin. Similarly, WO2009/149202 discloses cellulase variants exhibiting reduced affinity to lignin or ethanol or improved thermostability resulting from mutations at the equivalents of positions 63, 77, 129, 147, 153, 161, 194, 197, 203, 237, 247, 254, 281, 285, 289, 294, 327, 339, 344, 356, 378 and 382 in the linker peptide and catalytic domain of TrCel6A.